Structure of Lampbrush Chromosome Loops During Different States of Transcriptional Activity As Visualized in the Presence

Biology of the Cell, 59 (1987) 33-42 33 © Elsevier, Paris Original article Structure of lampbrush chromosome loops during different states of transcriptional activity as visualized in the presence I of physiological salt concentrations l,. Ulrich SCHEER * Division of Membrane Biology and Biochemistry, Institute of Cell and Tumor Biology, German Cancer Research Center, D-6900 Heidelberg, F.R.G. (Received 11-7-1986; accepted 17-11-1986) Lampbrush chromosomes of amphibian oocytes were isolated in the presence of near-physiological salt concentrations, to preserve their native state, and studied by electron microscopy of ultrathin s~dions. The transcriptional state of the lampbrush chromosomes was experimentally modulated by incubating the oocytes for various time periods in medium containing actinomycin D. The observations show that the structure of the lateral loops changes rapidly in response to alterations in transcriptional activity. During decreasing transcriptional activity and reduced packing density of transcripts, the chromatin axis first condensed into nucleosomes and then into an approximately 30 nm thick higher order chromatin fiber. Packaging of the loop axis into supranucleosomal structures may contribute to the foreshortening and retraction of the loops observed during inhibition of transcription and in later stages of meiotic prophase. The increasing packing density of the DNA during the retraction process of the loops could also be visualized by immunofluorescence microscopy using antibodies to DNA. The dependence of the loop chromatin structure on transcriptional activity is discussed in relation to current views of mechanisms involved in gene activation. lampbrush chromosomes - chromatin structure - electron microscopy - immunofluorescence microscopy - DNA antibodies INTRODUCTION transcribing RN A polymerases the chromatin fiber is largely extended and nucleosomes are absent [13]. Seve al lines of evidence suggest that changes in However, we also noted that in stages of reduced chromatin structure are involved in the transcriptional activity, when transcriptional com transcriptional regulation of gene expression (for plexes are more widely spaced on the chromatin axis, details see refs. [23, 31 , 42]). Nevertheless, the nucleosomal particles do appear in the immediate relationship between chromatin structure and vicinity of the RN A polymerase. This suggested that transcription is still poorly understood. Differences nucleosomes unfold anterior to transcribing between transcriptionally active and inactive polymerases and rapidly reform after each chromatin have been mainly established at the transcriptional event [34]. The rapid reformation of primary level of chromatin packaging,. the nucleosomes after the passage of a polymerase seems nucleosomes. Combined structural and biochemical to be a general feature of non-nucleolar chromatin observations indicate that nucleosomes are highly [e.g., 12, 21, 24, 34; for further refs. see 23, 30, 31]. dynamic structures which undergo reversible Correspondingly, an altered nucleosomal conformational changes during transcription. This is organization of chromatin, lacking the canonical particularly evident with nucleolar chromatin, i.e. nucleosomal repeat, has been found in biochemical pre-r RNA genes [12, 13, 20, 34, 45], but is not as clear experiments, for example, by nuclease digestion of for RNA polymerase II-transcribed genes. transcriptionally active protein-coding genes such as Based on observations made with electron heat shock genes of Drosophila [22, 44], chicken microscopic spread preparations of highly active ovalbumin genes [4, 5] and Balbiani ring genes of non-nucleolar chromatin, such as lamp brush Chironomous [43] and by cross-linking of histones to chromosome loops, we came to the conclusion that specific DNA sequences [19]. _ in stages of maximum packing density of Much less is known about the next higher level of chromatin folding, i.e., the chick chromatin fiber * Present address: Institute of Zoology I, University of with a diameter of usually about 30 nm [for reviews Wurzburg, Rontgenring 10, 0-8700 Wurzburg, F .R.G. see 10, 17], and its relation to transcription. At 34 U. Scheer present it is not clear, for example, whether the gene activation involves the unfolding of the thick chromatin fiber into a more extended nucleofilament and whether structural changes accompanying activation precede or are the consequence of transcription. Unfortunately, the higher order organization of chromatin cannot be readily studied in electron microscopic spread preparations since the methodology involves the unravelling of chromatin by exposure to very low ionic strength solutions that are known to destabilize higher order packaging [32, 41, 45]. However, in a few cases chromatin can be isolated at physiological salt concentrations thereby allowing the identification of transcribed chromatin regions with the attached transcripts by electron microscopy. Lampbrush chromosome loops of amphibian oocytes represent such an example. As originally shown by Mott and Callan [26], lampbrush chromosomes can be isolated under near physiological conditions and studied by electron microscopy of ultrathin sections ('end-embedding procedure'). Using this method, Spring and Franke [40] concluded that the intensely transcribed chromatin of lamp brush chromosome loops is not compacted into nucleosomal and supranucleosomal particles. A similar absence of higher order structures has also been found in the actively transcribed regions of Balbiani ring genes of Chironomus salivary glands prepared in situ by conventional electron microscopy [ I, 29]. In addition, it was observed by Andersson et al. [2, 3] that the thick chromatin fiber rapidly forms after experimental inhibition of transcription of the Balbiani ring genes with the drug DRB. To study in greater detail how the formation ofthe higher order structures is related to the specific transcriptional activity of a given chromatin strand, I have examined the structural organization of loop chromatin of 'native' lampbrush chromosomes in states of maximal and reduced transcriptional activities. The activity of amphibian lamp brush chromosomes can be modulated by varying the exposure of oocytes in vitro to the drug actinomycin D (AMD) and monitored by light microscopy, using the extent of loop retraction as a measure of transcri ption [6, 18]. Earlier studies based on electron microscopic spread preparations have shown that AMD induces a premature release of the RNP transcripts from the loop axes, thus leading to a progressive decrease in their packing density along the loops [34]. FIGU RE I. - Progressive retraction of the lateral loops of Triturus lampbrush chromosomes after increasing exposure times of oocytes to AMD. The lamp brush chromosomes were isolated in the presence of physiological salt concentrations and photographed with an inverted microscope using phase contrast optics. a, control; b, I hr; c, 2 hr; d, 3 hr; e, 4 hr; f, 5 hr after AMD treatment (50 pg/ m!). When oocytes are transferred after AMD treatment (5 hr) to medium without the drug, loops reappear within 20 hr (g). All micrographs are magnified to the same scale. X 600; bar = 20 pm. Lampbrush chromatin 35 The observations made in this study support the (Carl Zeiss, Oberkochen, F.R.G.). For electron microscopy, concept of a highly dynamic nature of the chromatin specimens were fixed with glutaraldehyde followed by osmium tetroxide and flat-embedded in Epon essentially as described [26, fiber and suggest that it is the transcriptional process 40]. Electron micrographs were taken with a Zeiss EM 10. as such that transforms the higher order thick fiber into a more extended conformation. This property of the chromatin fiber may also provide an explanation Immunofluorescence microscopy for the apparent retraction of lateral loops which The hybridoma clone AK 30-10 producing monoclonal accompany decreasing transcriptional activities. antibodies (lgM) to DNA was the result of a fusion of cells of the mouse myeloma line Ag 8.653 with the spleen cells of a BALB/ c mouse that was previously immunized with the high salt and detergent-resistant material of a rat hepatoma. Immunoglobulins were purified from ascites fluid by gel filtration on Sephacryl S300 columns (Pharmacia, Uppsala, MATERIALS AND METHODS Sweden) as described [16]. The antibody has been shown to bind to double- and single-stranded DNA (from prokaryotes and Light- and electron microscopy eukaryotic nuclei and mitochondria) but not to RN A [25]. A detailed account on this antibody, including binding studies by The newts Triturus cristatus carnifex were reared in our competitive ELISA-tests with a variety of synthetic laboratory. A piece of an ovary was removed from an oligonucleotides and natural nucleic acids, will be presented anesthetized female and placed in modified Barth's medium [14]. elsewhere [38]. Actinomycin D (Serva, Heidelberg) was added at a final concentration of 50 pg/ ml. Lampbrush chromosomes were For immunofluorescence microscopy, chromosomes attached to manually isolated from mid-sized oocytes (0 .8-1.0 mm in covers lips were transferred to 70% ethanol (10 min) and then diameter) in a medium containing 83 mM KCI, 17 mM NaCI, into phosphate-buffered saline (PBS). Antibodies to DNA were 0.01 mM CaCl2, buffered with 10 mM Tris-HCI to pH 7.2. The added at a concentration of 10pg/ ml for 20 min at room nuclear envelope was manually removed, the nuclear contents temperature. Following several

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